wisp-1 is a wnt-1- and b-catenin- responsive...

WISP-1 is a Wnt-1- and b-catenin-responsive oncogeneLifeng Xu,1 Ryan B. Corcoran,1 James W. Welsh,2 Diane Pennica,2 and Arnold J. Levine1,3,4

1Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 USA; 2Department of MolecularOncology, Genentech Inc., South San Francisco, California 94080 USA

WISP-1 (Wnt-1 induced secreted protein 1) is a member of the CCN family of growth factors. This studyidentifies WISP-1 as a b-catenin-regulated gene that can contribute to tumorigenesis. The promoter of WISP-1was cloned and shown to be activated by both Wnt-1 and b-catenin expression. TCF/LEF sites played a minorrole, whereas the CREB site played an important role in this transcriptional activation. WISP-1 demonstratedoncogenic activities; overexpression of WISP-1 in normal rat kidney fibroblast cells (NRK-49F) inducedmorphological transformation, accelerated cell growth, and enhanced saturation density. Although these cellsdid not acquire anchorage-independent growth in soft agar, they readily formed tumors in nude mice,suggesting that appropriate cellular attachment is important for signaling oncogenic events downstream ofWISP-1.

[Key Words: WISP-1; Wnt-1; b-catenin; oncogene]

Received November 16, 1999; revised version accepted January 24, 2000.

The Wnt/Wingless signaling pathway has been exten-sively studied in the past several years (Kinzler and Vo-gelstein 1996; Cadigan and Nusse 1997). A key player inthe Wnt/Wingless signaling cascade is b-catenin, whoseconcentration increases in response to the Wnt-1 signal.Cytoplasmic levels of b-catenin are regulated strictly byits interactions with the adenomatous polyposis coli(APC) (Munemitsu et al. 1995), axin (Zeng et al. 1997;Behrens et al. 1998) and GSK-3b (Rubinfeld et al. 1996;Yost et al. 1996) proteins. Failure of this regulatory path-way results in stabilization and subsequent accumula-tion of b-catenin in the cytoplasm. The elevated levels ofb-catenin in the cytoplasm promote its interactions withmembers of the TCF/LEF DNA-binding family (Korineket al. 1997; Morin et al. 1997; Willert and Nusse 1998).The bipartite complexes of b-catenin and TCF/LEFtranslocate to the nucleus and activate transcription ofdownstream genes (van de Wetering et al. 1997). Becausethe interaction between b-catenin and TCF/LEF familymembers and subsequent transcriptional activation can-not account for the diverse developmental processesregulated by the Wnt/Wingless signaling pathway, thereare probably other Wnt/Wingless and b-catenin-respon-sive transcription factors. It has been shown recentlythat stabilized armadillo, the b-catenin homolog in Dro-sophila, can also interact with the zinc finger proteinTeashirt, which promotes teashirt phosphorylation and

accumulation in the nucleus in response to Wingless sig-naling (Gallet et al. 1998, 1999).

The Wnt/Wingless signaling pathway is not only de-velopmentally important but is also implicated in mul-tiple cancers. One member of the Wnt family, Wnt-1,was originally identified as an oncogene activated by theinsertion of mouse mammary tumor virus in virus-in-duced mammary adenocarcinomas (Nusse et al. 1984).Chain-termination mutations of APC occur in most hu-man colon carcinomas (Polakis 1997), and mutationsthat stabilize b-catenin have been discovered in coloncarcinomas, melanomas, pilomatricomas, and hepato-cellular carcinomas (Morin et al. 1997; Rubinfeld et al.1997; de La Coste et al. 1998; Chan et al. 1999). Muta-tions of these Wnt/Wingless signaling pathway compo-nents all have the same consequence, the accumulationof b-catenin in the cytoplasm. It is believed that throughan interaction with DNA-binding factors, increased lev-els of b-catenin induce uncontrolled activation of down-stream genes, some of which might be involved in tu-morigenesis (Gumbiner 1997; Nusse 1997; Peifer 1997).Cyclin D1 and c-myc are two oncogenes that have re-cently been suggested to be regulated by b-catenin (He etal. 1998; Shtutman et al. 1999; Tetsu and McCormick1999).

To identify other downstream genes in the Wnt/Wing-less signaling pathway, especially those that play a rolein tumor development and progression, suppression sub-tractive hybridization was carried out between themouse mammary epithelial cell line C57MG and Wnt-1-expressing C57MG cells. The C57MG cells were cho-sen because they can be partially transformed by Wnt-1

3Present address: Rockefeller University, New York, New York 10021USA.4Corresponding author.E-MAIL [email protected]; FAX (212) 327-8505.

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expression (Mason et al. 1992). Wnt-1 induced secretedprotein 1 (WISP-1) was identified as one of the differen-tially expressed genes (Pennica et al. 1998). Sequenceanalysis revealed that WISP-1 shares high sequence ho-mology with the CCN family of growth factors, a familythat has been implicated in playing a role in tumorigen-esis (Lau and Lam 1999). This family is composed ofCTGF (connective tissue growth factor), Cyr-61 (cys-teine-rich 61), and nov (nephroblastoma overexpressed)(Brigstock 1999). CTGF is a downstream effector of TGF-b(Igarashi et al. 1993; Grotendorst et al. 1996; Kothapalliet al. 1997), and it can stimulate cell growth, up-regulateextracellular matrix protein expression, and induce an-giogenesis (Frazier et al. 1996; Babic et al. 1999; Shimo etal. 1999). Cyr-61 is an extracellular matrix-associatedsignaling protein that can augment growth factor-in-duced DNA synthesis, stimulate cell proliferation, andpromote angiogenesis and tumor growth (Kireeva et al.1996; Babic et al. 1998). nov was originally identified atan integration site of the myeloblastosis associated virus(MAV-1). It is considered to be a proto-oncogene becausean amino-terminal-truncated nov induces morphologicaltransformation when expressed in chicken and rat em-bryonic fibroblasts (Joliot et al. 1992).

In this study, the promoter of the WISP-1 gene wasisolated and WISP-1 was identified as a target gene regu-lated by Wnt-1 and b-catenin. A CREB-binding site wasshown to be a novel site mediating the activation of

WISP-1 by b-catenin. Furthermore, characterization ofthe oncogenic activity of WISP-1 demonstrated thatWISP-1 might contribute to b-catenin-mediated tumori-genesis.

Results

Construction of b-catenin mutants

b-catenin is not only a transcriptional activator in theWnt signaling pathway, but also a component of ad-herens junctions (Miller and Moon 1996). To study do-mains required for activation of b-catenin downstreamgenes, as well as the effect of b-catenin subcellularlocalization on the activation of these genes, four b-catenin mutants were constructed (Fig. 1A). Mutant4145 had both Thr-41 and Ser-45 at the amino-terminalGSK-3b phosphorylation site (Yost et al. 1996) changedto alanines. These changes resulted in a more stable formof b-catenin as demonstrated by pulse-chase analysis(data not shown). Mutant 4145TV not only had the abovemutations, but also had an additional double-point mu-tation that changed Thr-120 and Val-122 to alanines atthe a-catenin-binding region (Aberle et al. 1996). Thesemutations abrogated the binding of b-catenin toa-catenin, as demonstrated by immunoprecipitation analy-sis (Fig. 1B). Mutant nuclear localization signals (NLSs)had an SV40 large T antigen NLS (Boulikas 1993) cloned

Figure 1. (A) Schematic of b-catenin mutants.(Stippled boxes) The armadillo repeats. (B) Immunopre-cipitation of b-catenin mutants in C57MG cells. Myc-tagged b-catenin mutants were transfected intoC57MG cells. Cells were then metabolically labeledwith 35S-methionine for 5 hr and transfected b-cateninwas immunoprecipitated with anti-myc antibody 9E10(CalBiochem). Signals were visualized by autoradiogra-phy. (C) Mutant NLSs localized in the nucleus insteadof cytoplasm. Mutant 4145 (a) and mutant NLS (b)were transfected into 293 cells. Immunofluorescencestaining was performed with anti-myc antibody as thefirst antibody, followed by sequential incubation withbiotinylated goat anti-mouse IgG and FITC-conjugatedstreptavidin (Jackson Immunoresearch Laboratories).(D) Response of TopFlash reporter to b-catenin mu-tants. The 293 cell were transfected with 0.5 µg ofTopFlash reporter together with 0.5 µg of empty pCS2/MT vector, or different b-catenin mutants. A total of 1µg of pCS2/MT plasmid DNA was used to make up thetotal DNA to 2 µg; 0.1 µg of CMV–b-gal was cotran-fected to normalize transfection efficiency.

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in-frame at the amino terminus of 4145TV mutant ofb-catenin. It was localized to the nucleus as shown byimmunofluorescence staining when transfected into hu-man embryonic kidney 293 cells (Fig. 1C). Mutant D(C)introduced a stop codon at the carboxy-terminal regionof mutant 4145 and resulted in a protein product thatwas 88 amino acids shorter than wild type. Each of thesemutants were transfected into 293 cells together with aTopFlash luciferase reporter (which measures b-catenintranscriptional activity; Korinek et al. 1997) to test theiractivity on a known downstream promoter element (Fig.1D). Mutant 4145, 4145TV, and NLSs were able to in-duce the TopFlash reporter with comparable high levels.Compared with stabilized b-catenin, Mutant D(C) wasreduced in its ability to induce the TopFlash reporterbecause it lacked the transcriptional activation domain(van de Wetering et al. 1997).

Elevation of WISP-1 mRNA in C57MG cells expressingmutant b-catenin

WISP-1 was identified as one of the genes that was dif-ferentially expressed between parental C57MG cells andWnt-1-expressing C57MG cells (Pennica et al. 1998). Be-cause b-catenin is downstream of Wnt-1 in the Wnt sig-naling pathway, it is possible that Wnt-1 modulatesWISP-1 transcription through b-catenin. To investigatethe effect of b-catenin overexpression on the endogenousWISP-1 gene, C57MG cell lines expressing either Wnt-1or stabilized b-catenin mutants were generated. C57MGcells were chosen because it was shown that Wnt-1 up-regulates the expression of WISP-1 in these cells (Pen-

nica et al. 1998). Northern blot analysis showed thatWISP-1 mRNA was at much higher levels in C57MGcells expressing Wnt-1, stabilized b-catenin mutants4145 or 4145TV (about fourfold higher than controlcells), when compared with the C57MG parental cells(Fig. 2A).

WISP-1 is a downstream target gene of Wnt-1and b-catenin

To determine whether Wnt-1 and b-catenin regulatethe transcription of the WISP-1 gene, a 5.9-kb humanWISP-1 promoter fragment was isolated and cloned up-stream of the pGL2 basic luciferase reporter. A co-cul-ture experiment in which Wnt-1 protein was providedin a paracrine manner was carried out to determinewhether Wnt-1 signaling directly regulates transcrip-tional activation of the WISP-1 promoter. Pools ofC57MG cells transfected with the empty pGL2 basicvector (pGL2B), the WISP-1 promoter pGL2 basic lucif-erase construct (pGL2B-WP), or the TopFlash luciferasereporter were co-cultured with either the parental quailfibrosarcoma QT6 cells or Wnt-1 constitutively express-ing QTWnt-1 cells (Parkin et al. 1993). Luciferase activ-ity was measured 48 hr later. The WISP-1 luciferase re-porter was induced in response to the Wnt-1 signal, andthe induction level was almost comparable with that ofthe TopFlash reporter (Fig. 2B). To determine whetherthe activation of the WISP-1 promoter by Wnt-1 wasmediated by b-catenin, the WISP-1 luciferase reporterwas cotransfected into 293 cells with different dosages ofb-catenin mutant plasmids and luciferase analysis was

Figure 2. (A) Northern blot analysis ofWISP-1 RNA in C57MG cells expressingmutant b-catenin or Wnt-1. Total RNA(10 µg) from the cells was loaded onto eachlane. The blot was hybridized with a full-length mouse WISP-1 probe. For loadingcontrol, the blot was rehybridized with aprobe for mouse GAPDH gene. (B) Re-sponse of the WISP-1 promoter luciferasereporter (pGL2B-WP, WISP-1 sequence−5882 ∼−42, translation start site as +1) toWnt-1. C57MG cells were cotransfectedwith 1 µg of pGK-hygro and 10 µg ofpGL2B, pGL2B-WP, or TopFlash. Twodays after transfection, hygromycin wasadded to the medium for selection. Resis-tant colonies were pooled as stable celllines. In the coculture experiments, QT6(solid bars) and QTWnt-1 (open bars) cellswere plated on 10-cm plates at a density sothat after 48 hr they would be ∼40% con-fluent. The sample numbers of pGL2B,

pGL2B-WP, or TopFlash-transfected C57MG cells were then plated on tissue culture dishes containing either QT6 or QTWnt-1 cells.Luciferase analyses were carried out 48 hr after coculture. (C) WISP-1 promoter response to increasing amounts of b-catenin. The 293cell were transfected with 0.5 µg, 1.0 µg, or 1.5 µg of empty pCS2/MT vector or different b-catenin mutants together with 0.5 µg ofpGL2B-WP. A total of 0.1 µg of CMV–b-gal reporter was cotransfected with each sample to normalize transfection efficiency. EmptypCS2/MT vector was added to transfections to make a total of 2 µg of DNA. Luciferase analysis was carried out 60 hr after transfection.All luciferase measurements are expressed as means ±S.D. of triplicate cultures.

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carried out. The 293 cells were chosen because they hadmuch higher transfection efficiency than C57MG cellsand showed a much more sensitive response to b-cateninin TopFlash reporter assays (data not shown). Figure 2Cshows that the WISP-1 luciferase reporter was stronglytranscribed in response to b-catenin, especially stabi-lized b-catenin mutants 4145 and 4145TV, in a dose-dependent manner. Adding TCF to the transfection doesnot further increase the activation of the reporter (datanot shown). The carboxyl terminus of b-catenin ap-peared to be important in mediating this activation, asthe D(C) mutant was significantly reduced in its abilityto activate the WISP-1 reporter. Interestingly, we alsoobserved that although mutant NLS was able to induceTopFlash luciferase reporter to comparable levels as4145 and 4145TV, it failed to induce the WISP-1 lucifer-ase reporter in this analysis, suggesting that cytoplasmic

accumulation of b-catenin is important for activatingthe WISP-1 promoter.

The role of TCF/LEF sites and the CREB sitein the activation of the WISP-1 promoter by b-catenin

DNA-binding factors of the TCF/LEF family are knownto interact with b-catenin and activate many down-stream genes (Behrens et al. 1996; Molenaar et al. 1996).Therefore, a sequence analysis was carried out with theWISP-1 promoter to search for potential TCF/LEF-bind-ing consensus sequences. As depicted in Figure 3A, fiveputative TCF/LEF-binding sites were found in theWISP-1 promoter. Sites 1, 2, and 5 are imperfect matchesto the consensus TCF/LEF-binding sequence that differfrom the TCF/LEF core consensus sequence 58-CTTTGA/TA/T-38 at the 38 most base pair. Sites 3 and 4, on the

Figure 3. (A) Schematic representation of WISP-1 promoter pGL2 basic luciferase reporter constructs and deletion mutants. PutativeTCF/LEF (stippled boxes) and CREB (black boxes) binding sites are indicated. Their sequences are as follows; site 1 −4882 58-CACAAAG-38 −4876, site 2 −4677 58-CTCAAAG-38 −4671, site 3 −2978 58-CTTTGAT-38 −2972, site 4 −2546 58-CTTTGTT-38 −2540,site 5 −461 58-CTCAAAG-38 −455, CREB site −155 58-TGACGTCA-38 −148. (B) Response of WISP-1 promoter deletion contructs tob-catenin. The 293 cells were transfected with 0.5 µg of WISP-1 promoter, different deletion constructs, or empty pGL2 basic luciferasereporters, together with 1 µg of empty pCS2/MT vector, wild type, or 4145 b-catenin. A total of 0.1 µg of CMV–b-gal was cotransfectedto normalize transfection efficiency. (C) Schematic representation of Frag3 and TCF/LEF site mutation reporter constructs. Mutatedsites are as indicated. Transfections were carried out as described in B. (D) Schematic representation of Frag3, multiple TCF/LEF sitemutations, or CREB site mutation. Transfections were carried out as described in B.

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other hand, conform to the consensus sequence. To iden-tify the sequences in the WISP-1 promoter that mediatetranscriptional activation by b-catenin, a series of 58 pro-moter deletions were constructed (Fig. 3A). Each of thesedeletion constructs contained one less TCF/LEF consen-sus-binding site. Deletion mapping shows that deletingsequences containing TCF/LEF site 3 (Frag2), site 4(Frag1), or site 5 (Frag0), but not site 1 (Frag4) and site 2(Frag3), leads to consecutively reduced response of theWISP-1 promoter reporter to b-catenin (Fig. 3B). How-ever, these deletions are all large and not only eliminatethe TCF/LEF sites but also delete long stretches of sur-rounding sequences. To investigate the functional sig-nificance of the three promoter proximal TCF/LEF sitesfor the activation of the WISP-1 promoter, multiplepoint mutations were introduced into Frag3 that shouldrender each of these sites inactive (Brannon et al. 1997)(Fig. 3C). Frag3 was chosen for further study because itsresponse to b-catenin is comparable with that of the full-length promoter. Surprisingly, mutations in any of thesesites did not significantly affect the response of theWISP-1 promoter to b-catenin. Because functional re-dundancy of TCF/LEF sites had been reported (Brannonet al. 1997), multiple TCF/LEF sites were also elimi-nated by point mutations and their effect on the WISP-1promoter were tested. Knockout of multiple sites did notsignificantly affect the transactivation of the WISP-1promoter by b-catenin to any higher degree than thesingle-site mutations (Fig. 3D).

To further determine whether b-catenin activation ofthe WISP-1 promoter depends on TCF, a dominant-nega-tive form of TCF, DN–TCF (Korinek et al. 1997), wascotransfected into 293 cells with b-catenin. Consistentwith the results of site-directed mutagenesis experi-ments, DN–TCF had no significant effect on the trans-activation of the WISP-1 promoter by b-catenin (Fig. 4A),although it significantly reduced the activation of theTopFlash reporter by b-catenin in the positive controlexperiment (Fig. 4B).

Sequence analysis showed that there was a CREB-binding site at the 38 end of the WISP-1 promoter (Fig.3A). A CREB site has been reported to collaborate with aLEF site to regulate the transcription of TCR-a (Giese etal. 1995). Therefore, elimination of the CREB site wascarried out to evaluate the effect of this site on b-catenin-mediated transactivation. Surprisingly, knockout of theCREB site (Frag3-C1) not only reduced the basal activityof the WISP-1 promoter, but also reduced the activationby b-catenin from 9- to 10-fold to ∼5-fold (Fig. 3D). Thetransfections were repeated multiple times, each time intriplicate, and the CREB site elimination consistentlyreduced the response of the WISP-1 promoter to b-catenin. Frag3 with a deletion of 150 bp around the CREBsite also yielded similar results (data not shown). Thecombination of a CREB site mutation with TCF/LEF sitemutations did not further reduce the response of theWISP-1 promoter to b-catenin (data not shown), suggest-ing that CREB did not function through TCF/LEF.

To further investigate whether activation of theWISP-1 promoter by b-catenin depends on CREB protein,

a dominant-negative form of CREB, KCREB (Waltonet al. 1992), was cotransfected into 293 cells withb-catenin. As shown in Figure 5A, KCREB reducedb-catenin activation of the WISP-1 promoter to approxi-mately half of its original level. KCREB did not changethe response of TopFlash to b-catenin in the control ex-periment (Fig. 5B), suggesting that it did not suppressgeneral transcription. Thus, this study not only identi-fied WISP-1 as a downstream target gene transcription-ally activated by b-catenin, but also showed that TCF/LEF sites play only a minor role, whereas the CREB siteplays an important role in mediating the observedb-catenin-dependent transcriptional activation of WISP-1.

The oncogenic activity of WISP-1

WISP-1 shares high-sequence homology with growth fac-tors of the CCN family, which are involved in growthcontrol, embryonic development, and tumor formation(Brigstock 1999). CTGF and Cyr-61, two members of theCCN family, have mitogenic effects on some cell lines(Frazier et al. 1996; Kireeva et al. 1996). Therefore,experiments were carried out to determine whetherWISP-1 could also act as a mitogen. Human WISP-1(hWISP-1) protein was expressed by a baculovirus vector,

Figure 4. (A) Effects of dominant-negative TCF on the WISP-1promoter reporter. The 293 cells were transfected with 0.5 µg ofpCS2/MT, wild type, 4145, or 4145TV b-catenin, and 1.0 µg ofpcDNA3 (left) or DN–TCF (right), together with 0.5 µg ofWISP-1 promoter pGL2 basic luciferase reporter. A total of 0.1µg of CMV–b-gal was cotransfected to normalize transfectionefficiency. (B) Effects of dominant-negative TCF on the Top-Flash reporter. Transfections were carried out as described in Aexcept that 0.5 µg of TopFlash reporter was used.






purified from infected cell cultures, and tested for itsmitogenic activity on normal rat kidney fibroblast(NRK-49F) cells. Different concentrations of hWISP-1protein were added to plated NRK-49F cells. A dose-de-pendent stimulation of thymidine incorporation wasseen when cells were treated with a minimum concen-tration of 1.5 nM of hWISP-1 for 24 hr (Fig. 6), suggestingthat WISP-1 could promote cell proliferation. A three tofivefold increase in the rate of DNA synthesis was alsoseen in normal mouse lung (MLg) cells and mouse renaladenocarcinoma (RAG) cells (data not shown).

The ability of WISP-1 to support long-term growth ofcultured cells was then investigated. The hWISP-1cDNA was cloned into the retroviral expression vectorpBabe-puro. Retroviruses were made with the BOSC23packaging cell line (Pear et al. 1993) and used to infectseveral established mouse or rat cell lines. Puromycinwas added to infected cultures and resistant clones (>105)were pooled to generate hWISP-1-expressing cell linesand vector-expressing control cell lines. We found thatNRK-49F responded to WISP-1 overexpression by under-going changes in morphology and growth. Figure 7Ashows the expression of endogenous WISP-1 mRNA aswell as the expression of infected hWISP-1 mRNA inNRK cells. The biological responses to WISP-1 overex-pression in NRK cells were subsequently characterized.

Upon WISP-1 overexpression, NRK cells exhibitedprogressive morphological changes; they packed moredensely, adopted a more refractile appearance, and tendedto line up in uniform direction (Fig. 7B). An examinationof saturation density showed a threefold increase incells/cm2 on the culture dish surface compared with thecontrol cells (data not shown). WISP-1 overexpressionalso accelerated NRK cell growth. When cells wereplated at a density of 1 × 105 in DMEM supplementedwith 10% FBS, there was an approximately twofold dif-ference in growth rate between NRK–WISP1 and NRK–Vector cells. When grown in DMEM supplemented with0.5% FBS, the difference in growth rate was more dra-matic (Fig. 7C). Two WISP-1-expressing and two vector-expressing NRK cell lines were established via indepen-dent infections. Similar phenotypic alterations andgrowth rate accelerations were observed in both cases.

To assess the effect of WISP-1 overexpression on thetumorigenicity of NRK cells, the ability of NRK–vectorand NRK–WISP1 cells to form colonies in soft agar, aswell as their ability to form tumors in nude mice wasevaluated. The NRK–WISP1 cells from two independentinfections were tested. Experiments to determine the an-chorage-independent growth of these cells in soft agardemonstrated that although 293 control cells formedprominent colonies after 2 weeks, neither NRK–Vectornor NRK–WISP1 cells (line 1 and line 2) formed coloniesafter 6 weeks (data not shown). To test the capability ofthese cells to form tumors in nude mice, they were in-jected subcutaneously into 8-week-old nude mice andtumor growth was monitored every other week. Tumorsfirst became apparent in mice injected with NRK–WISP1cells (line 1 and line 2) 3 weeks after injection and grewrapidly. Twelve weeks after injection, all of the mice

Figure 5. (A) Effects of dominant-negative CREB on theWISP-1 promoter reporter. The 293 cells were transfected with0.5 µg of pCS2/MT, wild type, 4145, or 4145TV b-catenin, and1.0 µg of pcDNA3 (left) or KCREB (right), together with 0.5 µg ofWISP-1 promoter pGL2 basic luciferase reporter. A total of 0.1µg of CMV–b-gal was cotransfected to normalize transfectionefficiency. (B) Effects of dominant-negative CREB on the Top-Flash reporter. A total of 1.0 µg of pcDNA3 (left) or KCREB(right) was used. Transfections were carried out as described inA except that 0.5 µg of TopFlash reporter was used.

Figure 6. Effect of WISP-1 on DNA synthesis. NRK-49F cellswere plated in 96-well plates at 3 × 104 in HGDMEM with 10%serum. Twenty-four hours after plating, the medium waschanged to HGDMEM with 0.2% serum. WISP-1 was seriallydiluted in fresh medium and added at the indicated concentra-tions in a total volume of 70 µl/well. After 18 hr incubation at37°C, 5 µCi/ml [3H]-thymidine was added for 5 hr. Cells wereharvested onto a GF/C filter using Packard’s 96-well Filtermate196 and counted on a top count, microplate scintillationcounter (Packard). Values are the means of triplicate wells. Theexperiment was repeated at least four times with similar re-sults.

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injected with NRK–WISP1 cells developed tumors (line 1∼8–15 mm, line 2 ∼14–28 mm in diameter), whereasthose injected with NRK–Vector cells showed no tumorgrowth (#1 mm) (Fig. 8A). Northern blot analysis on tu-mor RNA showed strong expression of hWISP-1 in thetumors (Fig. 8B).

Discussion

WISP-1 was originally identified as one of the genes ex-pressed at higher levels in Wnt-1-transformed C57MGcells when compared with parental C57MG cells (Pen-nica et al. 1998). This study has shown that the elevatedexpression level of WISP-1 is due to the fact that theWISP-1 promoter element responds transcriptionally toWnt-1 and b-catenin signaling. To determine whetherWISP-1 mRNA was up-regulated by Wnt-1 without theneed for any new protein synthesis, QT6 or QTWnt-1cells were co-cultured with C57MG cells in the presenceof cycloheximide. However, the cycloheximide treat-ment itself induces WISP-1 mRNA expression inC57MG cells (data not shown), eliminating this ap-proach. The WISP-1 promoter was strongly induced byb-catenin in a dose-dependent manner, which is consis-tent with the possibility that the WISP-1 promotermight be directly regulated by b-catenin. Both the car-boxyl terminus of b-catenin (i.e., the transcriptional ac-tivation domain) and the cytoplasmic localization ofb-catenin appeared to be important for the induction ofthe WISP-1 promoter.

Placing WISP-1 downstream of b-catenin could ex-plain the observation that in 25 human colon adenocar-cinomas examined, WISP-1 mRNA was overexpressed(2- to >25-fold in ∼80% of tumors) compared with normalmucosa from the same patient (Pennica et al. 1998). Be-

cause most human colon cancers have elevated levels ofb-catenin either caused by loss-of-function APC muta-tions or stabilized b-catenin mutations, elevated WISP-1RNA expression might be the result of enhancedb-catenin-mediated transcription.

One surprising finding of this study was that TCF/LEFfactors played only a minor role in the induction of theWISP-1 promoter. This fact was supported by two linesof evidence. The first is that mutations of all putativeTCF/LEF sites had little effect on the b-catenin-depen-dent induction of the WISP-1 promoter, and the secondis that a dominant-negative mutant of TCF failed to in-hibit this induction. Instead, a CREB-binding site wasidentified as a sequence that played an important role inthe activation of the WISP-1 promoter. Not only did mu-tations in the CREB site reduce the activation of thispromoter, but also a dominant-negative mutant of CREBsignificantly inhibited this activation.

On the basis of these results, a novel pathway throughwhich b-catenin regulates some of its downstream genesmay be proposed. In this pathway, accumulation ofb-catenin in the cytoplasm might cause an increase incAMP levels. Elevated levels of cAMP activate proteinkinase A (PKA), which phosphorylates the CREB proteinand induces transcription of downstream genes throughthe CREB-binding site. Precisely how b-catenin inducescAMP levels remains to be elucidated. It also remainspossible that b-catenin activates an alternative kinasethat phosphorylates the CREB protein. In contrast to theprevailing model in which nuclear localized b-catenin/TCF complex turns on downstream gene transcription,this model postulates that the cytoplasmic localizationof b-catenin leads to the activation of a kinase, becausethe nuclear localized b-catenin failed to induce tran-scription of the WISP-1 promoter (Fig. 2C).

Figure 7. (A) Expression of hWISP-1mRNA in NRK-49F cells. NRK-49F cellswere infected with retroviruses expressingempty pBabe-puro vector or pBabe-hWISP-1 and selected with puromycin forresistant colonies. Stable colonies (>105)were pooled. Northern blot analysis wascarried out with 10 µg of total RNA. Full-length mouse WISP-1 cDNA was used as aprobe. The blot was rehybridized with aprobe for mouse GAPDH gene. (B) WISP-1expression alters the morphology of NRK–49F fibroblasts. Phase contrast microscopyof polyclonal NRK–vector and NRK–WISP1 fibroblasts cell lines. (1,2) NRK–vector and NRK–WISP1 cells at the samepassage number after infection, respec-tively. (C) WISP-1 expression alters thegrowth of NRK–49F cells. NRK–vector (d)and NRK–WISP1 (m) cells (both are of pas-sage 5) were seeded at low density (105/10-cm plate) and monitored for growth. Cellnumbers are presented as means ±S.D. ofthree plates.






Recently, a zinc finger protein Teashirt was reportedto bind to Armadillo (Drosophila b-catenin homolog)upon Armadillo stabilization in the cytoplasm (Gallet etal. 1998, 1999). This binding promotes phosphorylationand nuclear accumulation of Teashirt, leading to en-hanced transcription of Teashirt-dependent downstreamgenes. Taken together, these data suggest that b-cateninis able to regulate downstream events through multiplefactors, in addition to the TCF/LEF family members.The heterogeneity of genetic elements regulated byWnt-1 and b-catenin also suggests the functional diver-sity of the Wnt-signaling pathway.

One of the most important findings of this study is thecharacterization of an oncogene, WISP-1, as a Wnt-1-regulated gene. Overexpression of WISP-1 induced mor-phological transformation, increased cellular saturationdensity, and promoted growth in NRK-49F fibroblastcells in culture. The cells that overexpress WISP-1 pro-tein formed tumors in nude mice but not colonies in softagar, suggesting that appropriate cell attachment and ex-tracellular matrix formation might be important forthese cells to respond to WISP-1 and become tumori-

genic. Considering that CTGF and Cyr-61 are closelyrelated to WISP-1 and both signal through integrins(Kireeva et al. 1998; Babic et al. 1999), it is possible thatWISP-1 also triggers downstream events via integrin sig-naling. It remains to be elucidated whether tumor devel-opment in these nude mice is dependent on proper integ-rin binding.

The murine WISP-1 was found independently inmouse melanoma cells with a lower metastatic ability(Hashimoto et al. 1998), and called Elm1. Thus, this pro-tein has been associated with tumors in both the murineand human systems (Pennica et al. 1998). It is of interestthat WISP-1 can suppress metastasis in one melanomacell line (Hashimoto et al. 1998) and promote transfor-mation in the experimental system studied here. Thisindicates a complexity that needs to be studied further.

Mammary tumors from Wnt-1 transgenic mice havebeen investigated to study WISP-1 RNA expression pat-terns (Pennica et al. 1998). In situ hybridization revealedstrong staining in surrounding stromal cells with onlyfocal staining in epithelial cells. The high level ofWISP-1 RNA expression in the stroma surrounding thecarcinoma (epithelial cells) suggests that WISP-1 mightbe a stromal factor that exerts its effect in a paracrinemanner. The low level of focal expression in epithelialcells suggests that WISP-1 might also have an autocrineeffect. It has long been suggested that stromal factorsplay an important role during mammary tumor develop-ment and progression (Kanazawa and Hosick 1992;Hornby and Cullen 1995; Frazier and Grotendorst 1997;Sasaki et al. 1998). On the basis of these RNA expressionpatterns of WISP-1 in mammary tumors induced byWnt-1 expression, a model in which the paracrine andautocrine signals of WISP-1 contribute to the develop-ment of mammary tumors in Wnt-1 transgenic mice isproposed (Fig. 9). In this model, epithelial cells secreteWnt-1 protein, which then activates the Wnt-signalingpathway in surrounding stromal cells and leads to over-production of WISP-1. WISP-1 could stimulate the mam-

Figure 8. (A) WISP-1 expression induces tumorigenic potentialof NRK–49F cells. NRK–vector (passage 10) and NRK–WISP1(line 1, passage 6; line 2, passage 10; line 1 and line 2 are estab-lished from independent infections) were injected into nudemice (5 × 106) subcutaneously. None of the nude mice injectedwith NRK–vector (0/3) developed tumors, whereas all mice in-jected with either of the two lines of NRK–WISP1 (5/5 for eachline) had prominent tumor growth after 12 weeks of observa-tion. Tumors first became apparent 3 weeks after injection. (l)Vector; (j) WISP (line 1); (m) WISP (line 2). (B) WISP-1 mRNAexpression of tumor samples harvested from nude mice. North-ern blot analyses were carried out as described in Fig. 6. Tumors1 and 2 are representative tumors from NRK–WISP1 line 1 andline 2, respectively.

Figure 9. Putative model of WISP-1 paracrine and autocrinesignaling in mammary tumors of Wnt-1 transgenic mice.

Xu et al.





mary epithelia to proliferate in a paracrine manner. Fur-ther mutations accumulated during the uncontrolledproliferation of epithelial cells could eventually give riseto cells that constitutively produce WISP-1 and drive celldivision in an autocrine manner. It is of interest thatsome human colon carcinoma cell lines and human co-lon adenocarcinomas have WISP-1 gene amplifications(two to fourfold) (Pennica et al. 1998). Thus, a combina-tion of first a paracrine and then an autocrine productionof WISP-1 might efficiently stimulate epithelial cells tocontinue unregulated proliferation and eventual tumorformation.

Materials and methods

Sequences

The GenBank accession number for the hWISP-1 promoter re-gion is AF223404.

Cell cultures and DNA transfections

C57MG, 293, and NRK-49F cell lines were maintained inDMEM supplemented with 10% FBS. The quail fibrosarcomacell line QT6 and QTWnt-1 were obtained from Harold Varmus(NIH, Bethesda, MD). QTWnt-1 was maintained in mediumcontaining 400 µg/ml Geneticin.

The Wnt-1-expressing C57MG cell line was established byretroviral infections (Pennica et al. 1998). Cells were collected 6days after infection. Total RNA was extracted for Northern blotanalysis to assess WISP-1 mRNA expression. b-Catenin mu-tants expressing C57MG cell lines were established by trans-fecting C57MG cells with 4145 or 4145TV b-catenin plasmidstogether with pGK-hygro, a plasmid carrying the hygromycinresistance gene. Resistant colonies were selected and propa-gated in the presence of hygromycin. Two stable clones thatexpress 4145 or 4145TV at high levels were obtained. Cells werecollected and total RNA extracted for Northern blot analysis toassess WISP-1 mRNA expression. All DNA transfections wereperformed using the lipofectamine reagent (GIBCO BRL) as di-rected by the manufacturer.

Construction of b-catenin mutants

Myc-epitope-tagged wild-type Xenopus b-catenin in the pCS2/MT expression vector and empty pCS2/MT vector were pro-vided by Barry Gumbiner (Memorial Sloan Kettering CancerCenter, New York, NY). Site-directed mutagenesis experimentsto make b-catenin mutants 4145 and 4145TV were carried outas described previously (Lin et al. 1994). To make b-catenin NLSmutant, SV40 large T antigen NLS was subcloned in-frame infront of the myc tag repeats. To make D(C) mutant, PCR-basedsite-directed mutagenesis was used to generate a stop codon infront of the b-catenin internal PstI site. All mutants were se-quenced to confirm that only the intended point mutationswere introduced.

Northern blot analysis

Total RNA was isolated with the RNeasy Mini Kit (Qiagen) asdirected by the manufacturer. A total of 10 µg of RNA wasloaded onto 1.2% agarose gel containing 6% formaldehyde andrun in 1× E buffer (18 mM Na2HPO4 plus 2 mM NaH2PO4) over-night while circulating buffer. RNA was transferred to Nytranmembrane (Schleicher & Schuell) using the Turboblotter Trans-

fer System (Schleicher & Schuell). After UV cross-linking, blotswere prehybridized in Church buffer (0.2 M NaH2PO4, 0.3 M

Na2HPO4, 1% BSA, 7% SDS, 1 mM EDTA) for 2 hr at 65°C. Theprobes were labeled with [a32P]dCTP using the Prime-It RmTKit (Stratagene). Blots were hybridized with labeled probe over-night in Church buffer at 65°C and washed four times at 30 minin 1× SSC/0.1% SDS at 65°C before exposing to X-Omat film.

Isolation of genomic clones containing the WISP-1 promoter

The human genomic clone for WISP-1 was obtained using Ge-nome Systems, Inc. PCR-based DOWN TO THE WELL screen-ing method, according to the manufacturer’s instructions. Ge-nomic DNA was digested with BamHI and tested for hybridiza-tion to the hWISP-1 amino-terminal sequences by Southern blotanalysis. One hybridizing fragment, corresponding to −5882∼−42bp (numbered according to translation start site as +1) of theWISP-1 promoter, was gel purified, subcloned into the pBlue-script vector (Stratagene), and sequenced.

Construction of luciferase reporter plasmids

To clone the isolated WISP-1 promoter fragment to pGL2B, −5.9kb/pBlue-script construct was cut at the SmaI and SpeI sites onpBlue-script vector and cloned directionally into the SmaI andNheI sites of pGL2B. The 58 deletions of WISP-1 were generatedby digesting the −5.9 kb/pBlue-script construct at internal NheI(Frag4), KpnI (Frag3), XhoI (Frag2), and SacI (Frag1) sites andcloned directionally into pGL2B. To generate Frag0, PCR-basedsite-directed mutagenesis was carried out to introduce a KpnIsite at −328, then the PCR fragment was cut by KpnI and cloneddirectionally into pGL2B.

Site-directed mutagenesis was performed with theQuikChange Site-Directed Mutagenesis Kit (Stratagene). Themismatched oligonucleotides used to eliminate TCF sites 3–5are as follows: site 3, 58-GGTGGAATGGTCAGCGAATTCAT-CATGTTTTGGGGTGCC-38; site 4, 58-GGCCTCCTAGGATT-TATTTTTTGCTAGCTTTGTTTGCTTGTTTTG-38; site 5, 58-CACTGGGAGCCCTCTGCTAGCCCACACACCCGCCTG-38.The mismatched oligonucleotides used to eliminate CREB siteis as follows: 58-GGGTTTGTCCTTCACCCTGAATTCAGAT-CTTGCTTTAATAAAACC-38. Each mutagenic oligonucleo-tide introduces a restriction site (underlined) that was used toassay for incorporation of the mutation. All constructs weresequenced to confirm that only the intended point mutationswere introduced.

Luciferase assay

TopFlash luciferase reporter and DN–TCF were provided byBert Vogelstein (Johns Hopkins School of Medicine, Baltimore,MD). KCREB was provided by Richard Goodman (OregonHealth Science Center). Promoter fragments were subclonedinto pGL2 basic luciferase reporter plasmid (Promega). Lucifer-ase analysis was performed with Dual-Light Kit (Tropix) accord-ing to the manufacturer’s directions. Measurements were car-ried out with TR717 microplate luminometer (Tropix). Lucifer-ase readout was always obtained from triplicate transfectionsand averaged.

Retroviral infections and cell culture

Retroviral expression vector pBabe-puro was used to constructand express full-length hWISP-1. Recombinant retroviruseswere generated by transfecting the retroviral constructs intoBOSC23 packaging cells with lipofectamine. Forty-eight hourspost-transfection, retrovirus-containing supernatant was col-lected. Target cell lines were infected at ∼40% confluence by






replacing the culture medium with retrovirus-containing super-natant, supplemented with 1 ml of FBS and 8 µg/ml Polybrene.Infecting medium was replaced by 10 ml of DMEM plus 10%FBS 4–6 hr later. At 48 hr post-infection, cells were split intomedium containing 2.5 µg/ml puromycin. Resistant cloneswere pooled and maintained in puromycin.

To measure growth in low serum, cells were seeded at 105/10-cm plate and allowed to attach in the presence of 10% serumovernight. The following day, cells were washed once with PBSand then cultured in DMEM plus 0.5% FBS. To count cells, cellswere dissociated by trypsinization and viable cells were countedafter Trypan blue staining.

Growth in soft agar of NRK cells was assayed by embedding4 × 105 cells in 0.3% Noble agar (DIFCO Laboratory) in DMEMplus 10% FBS, over a substratum of 0.5% Noble agar in DMEMplus 10% FBS. No significant colony formation was observedafter 6 weeks of incubation. As a positive control, 293 cellsformed visible colonies after 2 weeks of incubation. Nude miceinjections were carried out by subcutaneously injecting 5 × 106

cells on the back of 8-week-old female BALB/c nude mice.

Acknowlegments

We thank A.K. Teresky for her help with the nude mice tumorexperiments, and A. Casau and L.A. Taneyhill for helpful com-ments on the manuscript. This work was supported by a grantfrom the National Cancer Institute.

The publication costs of this article were defrayed in part bypayment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 USC section1734 solely to indicate this fact.

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10.1101/gad.14.5.585Access the most recent version at doi: 14:2000, Genes Dev.

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-catenin-responsive oncogeneβ is a Wnt-1- and WISP-1

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